Histochemical demonstration of carbonic anhydrase activity in the alimentary canal

Summary Carbonic anhydrase (CAH) activity was histochemically demonstrated in various parts of the alimentary canal of rat and in the stomach of man using the method of Waldeyer and Häusler (1959). The most intense histochemical reaction was observed in the parietal cells of the rat stomach, and reactions of decreasing intensity in the epithelial cells of the colon, appendix, jejunoileum, duodenum and oesophagus in the order mentioned. An intense reaction was also observed in the parietal cells of the stomach of man and a weak activity in the pyloric glands. After electrophoresis on cellulose acetate film the CAH activity in human and rat stomach mucosa showed one band with the same migration rate as the fastest moving band of the erythrocyte CAH.     

Electrophoretic and histochemical studies of carbonic anhydrase activity

Summary A simple method for separation of carbonic anhydrase activity into components by electrophoresis on cellulose acetate strips is described. With this method, using barbiturate buffer systems at various pH values, two main components of CAH in rat erythrocytes, and the splitting of each of these into two minor components were revealed. Two components were also observed in the CAH activity in kidney and lens homogenates, and one component in brain homogenate. A modification of Häusler's histochemical method for CAH was adapted for visualization of the electrophoretically separated bands. This rendered the evalution of the results easier than with the quantitative measurements alone. The quantitative measurement of CAH activity in electrophoretic strips corresponded with the degree of staining by the histochemical method. This among other facts supports the view of the specificity of the histochemical method used. Some examples of the histochemical staining pattern of the CAH activity in rat tissues are given.     

Enhanced Healing of Rat Calvarial Defects with MSCs Loaded on BMP-2 Releasing Chitosan/Alginate/Hydroxyapatite Scaffolds

In this study, we designed a chitosan/alginate/hydroxyapatite scaffold as a carrier for recombinant BMP-2 (CAH/B2), and evaluated the release kinetics of BMP-2. We evaluated the effect of the CAH/B2 scaffold on the viability and differentiation of bone marrow mesenchymal stem cells (MSCs) by scanning electron microscopy, MTS, ALP assay, alizarin-red staining and qRT-PCR. Moreover, MSCs were seeded on scaffolds and used in a 8 mm rat calvarial defect model. New bone formation was assessed by radiology, hematoxylin and eosin staining 12 weeks postoperatively. We found the release kinetics of BMP-2 from the CAH/B2 scaffold were delayed compared with those from collagen gel, which is widely used for BMP-2 delivery. The BMP-2 released from the scaffold increased MSC differentiation and did not show any cytotoxicity. MSCs exhibited greater ALP activity as well as stronger calcium mineral deposition, and the bone-related markers Col1α, osteopontin, and osteocalcin were upregulated. Analysis of in vivo bone formation showed that the CAH/B2 scaffold induced more bone formation than other groups. This study demonstrates that CAH/B2 scaffolds might be useful for delivering osteogenic BMP-2 protein and present a promising bone regeneration strategy.     

Suppressor cell regulation of the spontaneously induced in vitro secondary antibody response.

The cytotoxic effect of peripheral blood lymphocytes from patients with chronic active hepatitis (CAH) on Chang liver cells was studied using a chromium-51 release assay. Cytotoxic effector cell activity was unaffected or slightly enhanced after the removal of glass-adherent mononuclear cells, indicating that the major effector cell is not a classical monocyte. Lymphocytes from 12 of 18 patients with CAH were cytotoxic, and the cytotoxic cells were contained within T-cell-depleted fractions. Further studies revealed that these effector cells were K cells, not B cells. K cells were identified as Fs- and complement receptor-bearing cells without detectable surface immunoglobulin. Serial studies in CAH showed continuous cytotoxicity in contrast to transient cytotoxicity in acute viral hepatitis, suggesting that the cytotoxic activity of lymphocytes is responsible for persistence of the disease.     

Carbonic anhydrase cytochemistry in mitochondria-rich cells of salamander larvae gill epithelium as related to age and H+ and Na+ concentrations

Carbonic anhydrase (CAH) was localized in the mitochondria-rich cells (MRC) of 1-week-old salamander larvae gill epithelium, in both MRC and pavement cells of 6-week-old larvae, and in regenerated stems of previously amputated gills. CAH activity of the MRC was measured quantitatively using a microscope densitometric technique. Changes in CAH activity per cell and changes in the numbers of CAH-positive MRC were followed under different H+ and Na+ concentrations at the two age groups. CAH activity per cell increased with age, whereas the numbers of CAH-positive MRC dropped. CAH activity per cell in the 1-week-old age group reached maximal values at pH 7.4 and stayed relatively high in the more alkaline media. Moderate increases of Na+ concentrations had small but significant effects on increasing CAH activity of gill MRC. When taking into consideration not only the changes in cellular activity but also the changes in the number of CAH-positive cells under the different acclimation media, an activity index (ICAH) was calculated. Thus, the ICAH in the 1-week-old was found to be dependent on the decline of ambient H+ concentrations (expressed as increasing pH), reaching maximal effect at pH 8.0. On the other hand, raising the Na+ concentrations of the acclimation media to 110 and 220 mOsm/liter caused a maximal inhibition of tissue CAH activity as expressed by ICAH. In conclusion, it is suggested that salamander larvae gill MRC take part in the adaptation of the larvae to changing H+ concentrations of their milieu rather than in their adaptation to changes in its osmolality.     

Hyperinsulinemia of chronic active hepatitis: impaired insulin removal rather than pancreatic hypersecretion

Exaggerated insulin response to oral glucose was demonstrated in peripheral blood of patients with chronic hepatic diseases. High peripheral insulin levels may be the result of pancreatic hypersecretion or decreased hepatic removal of insulin. The simultaneous assay of insulin and C-Peptide concentrations in peripheral blood enables the determination of both beta-cell activity and hepatic fractional insulin extraction. We have measured peripheral insulin and C-Peptide levels during OGTT in a group of subjects with chronic active hepatitis (CAH). These subjects showed glucose levels and incremental areas significantly higher than controls, but still in the upper range of normality. Insulin response to oral glucose was significantly greater in CAH patients than in controls, whereas C-Peptide levels and areas were quite similar in the two groups. The C-Peptide to insulin molar ratios before and after glucose, and the relations between C-Peptide and insulin incremental areas were lower in CAH patients than in controls. We conclude that the peripheral hyperinsulinemia observed in subjects with CAH is due to diminished insulin removal by the diseased liver rather than pancreatic hypersecretion.     

Increases in enzyme levels during the formation of phenolic acids in carrot cell cultures

The levels of glutamic acid dehydrogenase (GDH), phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (CAH) and O-methyltransferase (OMT) were measured during the formation of phenolic acids in carrot cells in suspension culture. Caffeic, ferulic and p-hydroxybenzoic acids were always present as the culture proceded. Total content of these acids increased at the early logarithmic and linear phases. GDH showed high activity at the early logarithmic and stationary phases. PAL activity was much enhanced at the linear and stationary phases. CAH activity was found in actively growing cells, especially at the early and late logarithmic phases OMT behaved similarly to PAL. The increases in GDH and CAH might be responsible for the rapid synthesis of phenolic acid at the early logarithmic phase. The increase in phenolic acid at the linear phase would certainly be due to enhancements of both PAL and OMT. On the other hand, the accumulation of vanillic acid was observed in cells which were transferred and cultured on an agar medium, but not in cells in suspension culture. This accumulation is related to increases in OMT levels and also to changes in the degree of β-oxidation.     

The correlation of phenylalanine ammonia-lyase and cinnamic acid-hydroxylase activity changes in Jerusalem artichoke tuber tissues

Summary Phenylalanine ammonia-lyase (PAL) and cinnamic acid-hydroxylase (CAH) exhibit parallel activity changes in tissues of Jerusalem artichoke (Helianthus tuberosus L.) tubers during ageing and in vitro culture. The activity of both enzymes appears in the tissue only after slicing and can be stimulated by manganese. Inhibition of CAH, by the deprivation of the substrate O₂, leads to cinnamic acid accumulation and PAL inhibition, a process prevented by cycloheximide. It is propounded that the link between PAL and CAH activities is the result of changes of the cinnamic acid pool size produced by a change in the activity of CAH.     

Antibodies to hepatocyte membrane antigens in chronic liver disease: detection by immunofluorescence after Bouin's fixation

Using a modified indirect immunofluorescent (IF) technique in which cryostat tissue sections were fixed in Bouin's solution for ten minutes prior to reaction with sera under test, we have looked for antibodies to the hepatocyte membrane (HMA) in the sera of patients with chronic active hepatitis (CAH) and primary biliary cirrhosis (PBC). Samples were tested initially in parallel on unfixed and Bouin's-fixed rat composite blocks (kidney, stomach, liver) at a titer of 1:100 and those found to be positive were diluted further and reexamined. Conventional unfixed sections of rat composite block showed no liver membrane immunofluorescence although antinuclear (ANA), mitochondrial (AMA), and smooth muscle antibodies (SMA) were detected as anticipated. By contrast, prior Bouin's fixation abolished most of the fluorescence due to ANA, AMA and SMA but resulted in brilliant fluorescence of rat hepatocyte membranes in eleven of twelve patients with CAH (93%) and all ten patients with PBC. Only one of 22 normals (5%), one of 20 with collagen-vascular diseases (5%), and one of seven with nonimmunologic liver disease (14%) were positive for this hepatocyte membrane antibody. Bouin's fixation prior to IF is a simple technique which appears to alter the hepatocyte membrane so that HMA become detectable. The strong association of HMA with CAH and PBC suggests that this test might be of value and may contribute towards a further understanding of the pathogenesis of these conditions.     

A cephalosporin C acetylhydrolase is present in the cultures of Nocardia lactamdurans

Two protein bands with strong esterase activity are present in broths of Nocardia lactamdurans MA4213 cultures. One of them shows cephalosporin C acetylhydrolase (CAH) activity. This activity is maximal at 48 h of growth and shows a pattern of regulation slightly different from that of cephamycin production in medium supplemented with glucose (166 mM), glycerol (326 mM) or ammonium chloride (60 mM). The CAH activity was purified to homogeneity by DEAE-Sepharose ion-exchange, Sephadex G-75 gel filtration, and phenyl-Sepharose hydrophobic interaction chromatography. It showed a molecular mass of 72,100 Da. The N-terminus of the protein was determined and showed the amino acid sequence GGAAPGGPGAHPLWLPAGKD. The enzyme showed K _m values of 7.0 mM and 8.3 mM for cephalosporin C and 7-aminocephalosporanic acid respectively but was not active on cephamycin C. Received: 17 December 1999 / Received revision: 22 February 2000 / Accepted: 25 February 2000     

Electron microscopic demonstration of carbonic anhydrase activity in mouse liver cells

Summary Carbonic anhydrase (CAH) activity was demonstrated ultracytochemically in the mouse liver cells fixed with 1% glutaraldehyde buffered to pH 7.2 with 0.1 M cacodylate buffer containing 0.1 M sucrose and other aldehyde fixatives. After the fixed 25–40 section were incubated in Hansson's incubation medium containing 0.2 M sucrose, the cobalt phosphate formed by the action of CAH was converted to lead phosphate by immersing the incubated sections into 0.1% lead nitrate aqueous solution.The lead phosphate precipitate was observed very well on the plasma membrane of hepatocytes in Disse space and of endothelial cells or erythrocytes, and very slightly on the external coat of microvilli in bili canaliculi.In the tissues fixed with 4% formaldehyde, the deposits were found very barely on the microvilli in the space of Disse and the plasma membrane of the endothelial cells or the erythrocytes.As the -hydroxyadipaldehyde-fixed tissues showed the highest the CAH activity but had not a good preservation of morphology, this fixative is not suitable for the electron microscopic histochemistry of CAH.The tissue incubated in medium containing Diamox exhibited non-specific deposits in all over the cell, which were lost when the tissue was treated in Diamox solution before incubation.     

Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana

Background

The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells.

Methodology/Principal Findings

Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited.

Conclusions/Significance

We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.     

Molecular Basis for Barbed End Uncapping by CARMIL Homology Domain 3 of Mouse CARMIL-1

Capping protein (CP) is a ubiquitously expressed, 62-kDa heterodimer that binds the barbed end of the actin filament with ∼0.1 nm affinity to prevent further monomer addition. CARMIL is a multidomain protein, present from protozoa to mammals, that binds CP and is important for normal actin dynamics in vivo. The CARMIL CP binding site resides in its CAH3 domain (CARMIL homology domain 3) located at or near the protein''s C terminus. CAH3 binds CP with ∼1 nm affinity, resulting in a complex with weak capping activity (30–200 nm). Solution assays and single-molecule imaging show that CAH3 binds CP already present on the barbed end, causing a 300-fold increase in the dissociation rate of CP from the end (i.e. uncapping). Here we used nuclear magnetic resonance (NMR) to define the molecular interaction between the minimal CAH3 domain (CAH3a/b) of mouse CARMIL-1 and CP. Specifically, we show that the highly basic CAH3a subdomain is required for the high affinity interaction of CAH3 with a complementary “acidic groove” on CP opposite its actin-binding surface. This CAH3a-CP interaction orients the CAH3b subdomain, which we show is also required for potent anti-CP activity, directly adjacent to the basic patch of CP, shown previously to be required for CP association to and high affinity interaction with the barbed end. The importance of specific residue interactions between CP and CAH3a/b was confirmed by site-directed mutagenesis of both proteins. Together, these results offer a mechanistic explanation for the barbed end uncapping activity of CARMIL, and they identify the basic patch on CP as a crucial regulatory site.     

Prenatal hormones and childhood sex segregation: playmate and play style preferences in girls with congenital adrenal hyperplasia

We investigated playmate and play style preference in children with congenital adrenal hyperplasia (CAH) (26 females, 31 males) and their unaffected siblings (26 females, 17 males) using the Playmate and Play Style Preferences Structured Interview (PPPSI). Both unaffected boys and girls preferred same-sex playmates and sex-typical play styles. In the conflict condition where children chose between a same-sex playmate engaged in an other-sex activity or an other-sex playmate engaged in a same-sex activity, boys (both CAH and unaffected brothers) almost exclusively chose playmates based on the preferred play style of the playmate as opposed to the preferred gender label of the playmate. By contrast, unaffected girls used play style and gender label about equally when choosing playmates. Girls with CAH showed a pattern similar to that of boys: their playmate selections were more masculine than unaffected girls, they preferred a boy-typical play style and, in the conflict condition, chose playmates engaged in a masculine activity. These findings suggest that prenatal androgen exposure contributes to sex differences in playmate selection observed in typically developing children and that, among boys and girls exposed to high levels of androgens prenatally, play style preferences drive sex segregation in play.     

A new DNA diagnostic system for the detection of human <Emphasis Type="Italic">CYP21</Emphasis> gene mutations associated with adrenal cortex hyperplasia

Congenital Adrenal Hyperplasia (CAH) is one of the most widespread severe autosomal recessive hereditary diseases. CAH is caused by the impaired biosynthesis of the key human hormones cortisol and aldosterone and is accompanied by the excess synthesis of androgens. Over 90% of CAH cases are caused by a deficiency of the steroid 21-hydrohylase (P450c21). The degree of damage in this enzyme is responsible for the severity of the clinical manifestation of CAH from potentially lethal to mild symptoms. Various mutations of the gene encoding this enzyme are the main source of the reduced activity of the steroid-21-hydrolase. The location of the highly homological pseudogene CYP21P in close proximity to the functional gene impedes the DNA diagnostics of CAH. To detect the eight most frequent CYP21 gene mutations associated with CAH, we developed a new real-time PCR-based system of DNA diagnostics using new allele-specific primers and TaqMan probes for the analyzed mutations. The method was primarily tested on artificial DNA templates, where the analyzed mutations were introduced by site-directed mutagenesis. Then, it was tested on DNA samples from 43 patients with clinical and biochemical manifestations of CAH; seven patients were used as a control. Two mutant alleles were detected in two different individuals: the nonsense Q318X and the missense V281L mutations.     

HLA Typing of Patients with 21-Hydroxylase Deficiency in Iranian Children with Congenital Adrenal Hyperplasia

Congenital adrenal hyperplasia (CAH) is a group of potentially life-threatening disorders, most often caused by deficiency of steroid 21-hydroxylase. Children with ambiguous genitalia, hermaphroditism, or signs and symptoms of CAH admitted to Children's Medical Center were enrolled in the survey, and 101 patients were found. Karyotyping, clinical examination, and paraclinical tests were done. HLA typing was done in patients with proven classical CAH and their parents. HLA antigens were typed in children with CAH-type 21-hydroxylase deficiency. The antigen frequencies were compared with those of the control population. The studies revealed that two HLA antigens, HLA-B18 and HLA-B21, showed a significant increase in frequency. The calculated relative risk value was high, distinguishing the population of patients and their parents. The relative risk among patients was 11.82 for HLA-B18 and 1.75 for HLA-B21 antigens. There was no relationship between HLA-DR antigens and CAH. Studies on the correlation between HLA and CAH indicate an association with HLA-B18 and HLA-B21 antigens, and they can be used as genetic markers of the disorder in the Iranian population, if they are restricted to Iranian patients.     

Expression of the human genes for steroid 21-hydroxylase and its C169R mutant in insect cells and functional analysis of the expression products

Steroid 21-hydroxylase (CYP21A2) is a key enzyme of glucocorticoid and mineralocorticoid biosynthesis in the adrenal cortex and belongs to the family of microsomal cytochrome P450. CYP21A2 deficiency is the most common cause of human congenital adrenal hyperplasia (CAH). Human CYP21A2 and its C169R mutant, observed in a patient with classic CAH, were expressed in Sf9 and Hi5 insect cells infected with recombinant baculoviruses. Functional CYP21A2 was produced to 28% of the total microsomal protein under optimal conditions. The C169R mutation did not affect the efficiency of CYP21A2 synthesis in insect cells, nor did it prevent CYP21A2 incorporation in membranes of the endoplasmic reticulum. Functional analysis in vitro showed that the mutant enzyme almost completely lacked the catalytic activity towards two substrates, progesterone and 17-hydroxyprogesterone.     

Hepatitis B core antigen-specific IFN-gamma production of peripheral blood mononuclear cells in patients with chronic hepatitis B virus infection

To evaluate the specificity of cellular immune response to hepatitis B virus (HBV) Ag in patients with chronic HBV infection, we have measured IFN-gamma production and proliferation of PBMC of 16 patients with chronic active hepatitis (CAH), 17 asymptomatic carriers of HBV (ASC), 6 anti-hepatitis B surface (HBs)-positive subjects, and 6 control individuals with ELISA procedure and [3H]thymidine incorporation. There was no significant increase in the mean proliferative response to recombinant HB surface and core Ag (rHBsAg and rHBcAg), nor was IFN-gamma production elicited with rHBsAg in any group. In contrast, PBMC of HBeAg-positive and anti-HBe-positive CAH patients, and anti-hepatitis B "e" Ag (HBe)-positive ASC showed significantly enhanced IFN-gamma production in response to HBcAg, whereas those of HBeAg-positive ASC and anti-HBs-positive subjects did not respond to HBcAg. The maximal response was observed in a 5-day culture with 500 ng/ml of rHBcAg when assessed by stimulation index value. Monocytes did not demonstrate an increased suppressor or helper activity for IFN-gamma production in these patients. T cell subset fractionation revealed that CD4+ cells were main population of IFN-gamma production specific for HBcAg and CD8+ cells did not suppress IFN-gamma production of CD4+ cells. Furthermore, CD4+ cells of HBeAg-positive ASC generated lesser amounts of IFN-gamma than HBeAg-positive CAH patients did. These results show that the measurement of IFN-gamma production is useful to determine cellular immune response to HBV Ag and suggest that IFN-gamma production depends on the helper activity of CD4+ T cells sensitized to HBcAg.